Use of rapamycin derivatives in vasculopathies and xenotransplantation

ABSTRACT

Use of a rapamycin derivative of formula I as defined in the claims for preventing or treating vasculopathies and manifestations of xenotransplantation.

The present invention relates to a new use, in particular a new use fora compound group comprising derivatives of rapamycin, in free form or inpharmaceutically acceptable salt or complex form.

Suitable derivatives of rapamycin include e.g. compounds of formula I

wherein

X is (H,H) or O;

Y is (H,OH) or O;

R¹ and R² are independently selected from

-   -   H, alkyl, arylalkyl, hydroxyalkyl, dihydroxyalkyl,        hydroxyalkoxycarbonylalkyl, hydroxyalkylarylalkyl,        dihydroxyalkylarylalkyl, acyloxyalkyl, aminoalkyl,        alkylaminoalkyl, alkoxycarbonylaminoalkyl, acylaminoalkyl,        arylsulfonamidoalkyl, allyl, dihydroxyalkylallyl,        dioxolanylallyl, dialkyl-dioxolanylalkyl,        di(alkoxycarbonyl)-triazolyl-alkyl and hydroxy-alkoxy-alkyl;        wherein “alk-” or “alkyl” is C₁₋₆alkyl, branched or linear;        “aryl” is phenyl or tolyl; and acyl is a radical derived from a        carboxylic acid; and    -   R⁴ is methyl or    -   R⁴ and R¹ together form C₂₋₆alkyl;

provided that R¹ and R² are not both H; and hydroxyalkoxyalkyl is otherthan hydroxyalkoxymethyl.

Such compounds are disclosed in WO 94/09010 the contents of which, inparticular with respect to the compounds, are incorporated herein byreference.

Acyl as may be present in R₁ or R₂, is preferably R_(a)CO— wherein R_(a)is C₁₋₆alkyl, C₂₋₆alkenyl, C₁₋₆cycloalkyl, aryl, aryl C₁₋₆alkyl (whereinaryl is as defined above) or heteroaryl, e.g. a residue derived from a 5or 6 membered heterocycle comprising N, S or O as a heteroatom andoptionally one or two N as further heteroatoms. Suitable heteroarylinclude e.g. pyridyl, morphohno, piperazinyl and imidazolyl.

Examples of such compounds include:

1. 40-O-Benzyl-rapamycin

2. 40-O-(4′-Hydroxymethyl)benzyl-rapamycin

3. 40-O-[4′-(1,2-Dihydroxyethyl)]benzyl-rapamycin

4. 40-O-Allyl-rapamycin

5.40-O-[3′-(2,2-Dimethyl-1,3-dioxolan-4(S)-yl)-prop-2′-en-1′-yl]-rapamycin

6. (2′E, 4′S)-40-O-(4′,5′-Dihydroxypent-2′-en-1′-yl)-rapamycin

7. 40-O-(2-Hydroxy)ethoxycarbonylmethyl-rapamycin

8. 40-O-(2-Hydroxy)ethyl-rapamycin

9. 40-O-(3-Hydroxy)propyl-rapamycin

10. 40-O-(6-Hydroxy)hexyl-rapamycin

11. 40-O-[2-(2-Hydroxy)ethoxy]ethyl-rapamycin

12. 40-O-[(3S)-2,2-Dimethyldioxolan-3-yl]methyl-rapamycin

13. 40-O[(2S)-2,3-Dihydroxyprop-1-yl]-rapamycin

14. 40-O-(2-Acetoxy)ethyl-rapamycin

15. 40-O-(2-Nicotinoyloxy)ethyl-rapamycin

16. 40-O-[2-(N-Morpholino)acetoxy]ethyl-rapamycin

17. 40-O-(2-N-Imidazolylacetoxy)ethyl-rapamycin

18. 40-O-[2-(N-Methyl-N′-piperazinyl)acetoxy]ethyl-rapamycin

19. 39-O-Desmethyl-39,40-O,O-ethylene-rapamycin

20. (26R)-26-Dihydro-40-O-(2-hydroxy)ethyl-rapamycin

21. 28-O-Methyl-rapamycin

22. 40-O-(2-Aminoethyl)-rapamycin

23. 40-O-(2-Acetaminoethyl)-rapamycin

24. 40-O-(2-Nicotinamidoethyl)-rapamycin

25. 40-O-(2-(N-Methyl-imidazo-2′-ylcarboxamido)ethyl)-rapamycin

26. 40-O-(2-Ethoxycarbonylaminoethyl)-rapamycin

27. 40-O-(2-Tolylsulfonamidoethyl)-rapamycin

28.40-O-[2-(4′,5′-Dicarboethoxy-1′,2′,3′-triazol-1′-yl)-ethyl]-rapamycin

A preferred compound is e.g. 40-O-(2-hydroxy)ethyl-rapamycin (referredthereafter as Compound A).

Compounds of formula I have, on the basis of observed activity, e.g.binding to macrophilin-12 (also known as FK-506 binding protein orFKBP-12), e.g. as described in WO 94/09010. been found to be useful e.g.as immunosuppressants, e.g. in the treatment of acute allograftrejection.

Organ transplants of liver, kidney, lung and heart are now regularlyperformed as treatment for endstage organ disease. Because of thecurrent shortage of human donors for transplantable allografts,attention has focused on the possibility of using xenografts(transplants between species) in transplantation. One of the majorobstacles in transplanting successfully xenografts in humans isimmunological.

A further obstacle in allo- and xenotransplantation is the chronicrejection and thus organ transplantation is not yet a clinically viablesolution to irreversible organ disease.

Chronic rejection, which manifests as progressive and irreversible graftdysfunction, is the leading cause of organ transplant loss, in somecases already after the first postoperative year. The clinical problemof chronic rejection is clear from transplantation survival times; abouthalf of kidney allografts are lost within 5 years after transplantation,and a similar value is observed in patients with heart allografts.

Chronic rejection is considered as a multifactorial process in which notonly the immune reaction towards the graft but also the response of theblood vessel walls in the grafted organ to injury (“response-to-injury”reaction) plays a role. The variant of chronic rejection with the worstprognosis is an arteriosclerosis-like alteration, also called transplantvasculopathy, graft vessel disease, graft arteriosclerosis, transplantcoronary disease, etc. This vascular lesion is characterized bymigration and proliferation of smooth muscle cells, probably underinfluence of growth factors that are amongst others synthesized byendothelial cells. This leads to intimal proliferation and thickening,smooth muscle cell hypertrophy repair, and finally to gradual luminalobliteration (vascular remodelling). It appears to progress also throughrepetitive endothelial injury induced amongst others by host antibody orantigen-antibody complexes; also so-called non-immunological factorslike hypertension, hyperlipidemia, hypercholesterolemia etc. play arole.

Chronic rejection appears to be inexorable and uncontrollable becausethere is no known effective treatment or prevention modality. Thus,there continues to exist a need for a treatment effective in preventing,controlling or reversing manifestations of chronic graft vesseldiseases.

There also continues to exist a need to prevent or treat restenosis orvascular occlusions as a consequence of proliferation and migration ofintimal smooth muscle cell, e.g. induced by vascular surgeries such asangioplasty.

In accordance with the present invention, it has now surprisingly beenfound that compounds of formula I inhibit vasculopathies such asvascular remodelling and are particularly indicated to prevent or combatchronic rejection in a transplanted organ.

In accordance with the particular findings of the present invention,there is provided:

1. A method for preventing or treating neointimal proliferation andthickening in a subject in need thereof, comprising administering tosaid subject a therapeutically effective amount of a compound of formulaI.

In a series of further specific or alternative embodiments, the presentinvention also provides:

2.1. A method for preventing or combating manifestations of chronicrejection in a recipient of organ or tissue transplant comprising thestep of administering to said recipient a therapeutically effectiveamount of a compound of formula I.

2.2. A method for preventing or combating graft vessel diseases, e.g.transplant vasculopathies. arteriosclerosis or atherosclerosis, in arecipient of organ or tissue transplant. comprising the step ofadministering to said recipient a therapeutically effective amount of acompound of formula I.

-   -   By manifestations of chronic rejection are meant the conditions        resulting from the immune reaction towards the graft and the        response of the blood vessel walls in the grafted organ or        tissue as indicated above. Compounds of formula I are useful for        reducing chronic rejection manifestations or for ameliorating        the conditions resulting from chronic rejection.    -   The organ or tissue transplantation may be performed from a        donor to a recipient of a same or different species. Among such        transplanted organs or tissues and given illustratively are        heart, liver, kidney, spleen, lung, small bowel, and pancreas,        or a combination of any of the foregoing.

In a further or alternative embodiment the invention provides:

3. A method for preventing or treating intimal smooth muscle cellproliferation and migration, e.g. restenosis, and/or vascular occlusionfollowing vascular injury, e.g. angioplasty, in a subject in needthereof, comprising administering to said subject a therapeuticallyeffective amount of a compound of formula I.

In a further or alternative embodiment, the present invention alsoprovides:

4. A method for preventing or combating acute or chronic rejection in arecipient of organ or tissue xenograft transplant comprisingadministering to said recipient a therapeutically effective amount of acompound of formula I.

-   -   Xenograft organ or tissue transplants include e.g. heart, liver,        kidney, spleen, lung, small bowel, pancreatic (complete or        partial, e.g. Langerhans islets), skin and bone marrow        xenografts.

As alternative to the above the present invention also provides:

5. A compound of formula I for use in any method as defined under 1 to 4above; or

6. A compound of formula I for use in the preparation of apharmaceutical composition for use in any method as defined under 1 to 4above; or

7. A pharmaceutical composition for use in any method as defined under 1to 4 above comprising a compound of formula I together with one or morepharmaceutically acceptable diluents or carriers therefor.

Utility of the compounds of formula I in treating diseases andconditions as hereinabove specified, may be demonstrated in animaltests, for example in accordance with the methods hereinafter described.

A. Chronic Allograft Rejection

The kidney of a male DA (RT1^(a)) rat is orthotopically transplantedinto a male Lewis (RT1¹) recipient. In total 24 animals aretransplanted. All animals are treated with cyclosporine A at 7.5mg/kg/day per os for 14 days starting on the day of transplantation, toprevent acute cellular rejection. Contralateral nephrectomy is notperformed. Each experimental group treated with a distinct dose of acompound of formula I or placebo comprises six animals.

Starting at day 53-64 after transplantation, the recipient animals aretreated per os for another 69-72 days with a compound of formula I orreceive placebo. At 14 days after transplantation animals are subjectedto graft assessment by magnetic resonance imaging (MRI) with perfusionmeasurement of the kidneys (with comparison of the grafted kidney andthe own contralateral kidney). This is repeated at days 53-64 aftertransplantation and at the end of the experiment. The animals are thenautopsied. Rejection parameters such as MRI score, relative perfusionrate of the grafted kidney and histologic score of the kidney allograftfor cellular rejection and vessel changes are determined andstatistically analyzed. Administration of a compound of formula I, e.g.Compound A, at a dose of 0.5 to 2.5 mg/kg in this rat kidney allograftmodel yields a reduction in all above mentioned rejection parameters. Inthis assay, animals treated per os with 2.5 mg/kg/day of Compound A havea significantly lower MRI score of rejection, histologic score forcellular rejection and vessel changes and a significantly lowerreduction in perfusion rate assessed by MRI than the animals of theplacebo group.

B. Aorta Transplantation

In this model of aorta transplantation in the rat, an allogeneicresponse to the graft does not destroy the graft, but it evokespathological changes resembling those of chronic rejection in clinicaltransplantation. These include infiltration into the adventitia ofmononuclear cells (lymphocytes, macrophages, some plasma cells), andthickening of the intima.

Donor aorta between the branch of the renal artery and the start of thecaudal mesenteric aorta, about 1 cm in length, is harvested from a maleDA (RT1^(a)) rat and transplanted orthotopically in a male Lewis (RT1¹)rat. Weekly after transplantation, the body weight is recorded. Atautopsy, the graft with part of the aorta of the recipient just aboveand below the transplant is removed. It is perfused ex vivo withphosphate-buffered saline supplemented with 2% paraformaldehyde and 2.5%glutaraldehyde for about 2 minutes, then for 24 hours fixed by immersionfixation in the same solution, and thereafter fixed in 4% bufferedformalin. Pieces of the graft are embedded in paraffin. in such a waythat both a transversal section and a longitudinal section is made ofthe grafted aorta and the recipient's own aorta.

Sections of 4 μm thickness are stained by hematoxylin-eosin,elastica-von-Gieson and periodic-acid-Schiff. Apart from conventionallight microscopy, images are recorded by confocal laser scanningmicroscopy. From each section, four areas are scanned, and from eacharea the thickness of the intima and intima+media is measured at fivelocations.

At autopsy. weight and histology is performed for thymus, spleen, liver,kidney, testes and seminal vesicles.

A first experiment includes 4 groups, each comprising 4 animals. In onegroup isogeneic transplantations (Lewis to Lewis) are performed, andanimals receive a placebo microemulsion: the other groups compriseallogeneic transplantations, and animals receive per os either placebomicroemulsion or a compound of formula I in microemulsion at 2.5mg/kg/day. The experiment is terminated at 7 weeks aftertransplantation.

A second experiment includes 4 groups, each comprising 4 animals. In allcases allogeneic transplants are performed, and animals receive per oseither placebo microemulsion or a compound of formula I in microemulsionat 0.63, 1.25, 2.5 or 5.0 mg/kg/day. The experiment is terminated 11weeks after transplantation.

In both experiments, the compounds of formula I, particularly Compound Asignificantly inhibit graft infiltration and neointima formation.

C. Angioplasty

Studies on angioplasty are done in the model of balloon catheter injury:Balloon catheterization is performed on day 0, essentially as describedby Powell et al. (1989). Under Isofluorane anaesthesia, a Fogarty 2Fcatheter is introduced into the left common carotid artery via theexternal carotid and inflated (distension≈10 μl H2O). The inflatedballoon is withdrawn along the length of the common carotid three times,the latter two times whilst twisting gently to obtain a uniformde-endothelialization. The cathether is then removed, a ligature placedaround the external carotid to prevent bleeding and the animals allowedto recover.

2 groups of 12 RoRo rats (400 g, approximately 24 weeks old) are usedfor the study: one control group and one group receiving the compound offormula I. The rats are fully randomized during all handling,experimental procedures and analysis.

The compound to be tested is administered p.o. (gavage) starting 3 daysbefore balloon injury (day −3) until the end of the study, 14 days afterballoon injury (day +14). Rats are kept in individual cages and allowedfood and water ad libidum.

The rats are then anaesthetized with Isofluorane, a perfusion catheterinserted through the left ventricle and secured in the aortic arch, andan aspiration cannula inserted into the right ventricle. Animals areperfused under a perfusion pressure of 150 mmHg, firstly for 1 min. with0.1 M phosphate buffered saline solution (PBS, pH 7.4) and then for 15min. with 2.5% glutaraldehyde in phosphate buffer (pH 7.4). Theperfusion pressure is 150 mmHg at the tip of the cannula 100 mmHg in thecarotid artery), as determined in a preliminary experiment byintroducing a cannula attached to a pressure transducer into theexternal carotid). Carotid arteries are then excised, separated fromsurrounding tissue and immersed in 0.1 M cacodylate buffer (pH 7.4)containing 7% saccharose and incubated overnight at 4° C. The followingday the carotids are immersed and shaken for 1 h at room temperature in0.05% KMnO4 in 0.1 M cacodylate. The tissues are then dehydrated in agraded ethanol series; 2×10 min in 75%, 2×10 min in 85%, 3×10 min in 95%and 3×10 min in 100% ethanol. The dehydrated carotids are then embeddedin Technovit 7100 according to the manufacturers recommendation. Theembedding medium is left to polymerize overnight in an exsiccator underargon, since oxygen is found to inhibit proper hardening of the blocks.

Sections 1-2 μm thick are cut from the middle section of each carotidwith a hard metal knife on a rotary microtome and stained for 2 min withGiemsa stain. About 5 sections from each carotid are thus prepared andthe cross-sectional area of the media, neointima and the lumenmorphometrically evaluated by means of an image analysis system (MCID,Toronto, Canada).

In this assay, the compounds of formula I inhibit myointimalproliferation when administered per os at a daily dose of from 0.5 to2.5 mg/kg. Intimal thickening is significantly less in the vessels ofthe rats that receive Compound A compared to the control animals, e.g.at 0.5 mg/kg statistical inhibition of neointima formation of 50% at 2.5mg/kg significant inhibition of 75%.

D. In Vivo Heart Xenotransplantation (Hamster-to-Rat)

The hamster-into-rat xenograft combination is a so-called difficultconcordant combination. Rats do not have natural anti-hamster antibodyin sufficient amounts to yield immediate hyperacute rejection asobserved in concordant combinations; however, rejection in untreatedrecipients occurs within 3-4 days, by antibodies in combination withcomplement. This is visualized in histology by destruction of bloodvessels, exsudation and extravasation of erythrocytes, and influx bypolymorpho-nuclear granulocytes; often there are signs of hemorrhage andthrombosis. Once this rejection has been overcome by effectiveinhibition of antibody synthesis or complement inactivation, a cellularrejection can emerge later on. This is visualized in histology by influxof mononuclear cells, including lymphocytes, lymphoblastoid cells, andmacrophages, and destruction of the myocyte parenchyma. The inhibitionof cellular rejection requires more immuno-suppression than that ofallografts. Congenitally athymic (mu/mu) rats lack a competent(thymus-dependent) cellular immune system and generally are unable toreject allografts. Such animals do reject a hamster xenograft within 3-4days in a similar fashion as euthymic rats, indicative that (at leastpart of) anti-hamster antibody synthesis in rats occurs following athymus-independent B-cell response. Such recipients are useful inhamster xenografting to evaluate rejection by thymus-independentantibody-mediated rejection.

The heart of a Syrian hamster is heterotopically transplanted in theabdomen of a male Lewis (RT1′) rat with anastomoses between the donorand recipient's aorta and the donor right pulmonary artery to therecipient's inferior vena cava. The graft is monitored daily bypalpation of the abdomen. Rejection is concluded in case of cessation ofheart heat. Animals are weighed weekly. In the present series ofexperiments, the endpoint is set to 28 days. Animals are subjected toautopsy; apart from the graft, weight and histology is assessed forthymus, spleen, liver, seminal vesicles and testes. Blood is taken andprocessed to serum for the determination of cytolytic anti-hamstererythrocyte antibody and hemolytic complement activity.

In this assay, compounds of formula 1, e.g. Compound A, result inprolonged graft survival, in both athymic and euthymic recipients.

Daily dosages required in practicing the method of the present inventionwill vary depending upon, for example, the compound of formula Iemployed, the host, the mode of administration and the severity of thecondition to be treated. A preferred daily dosage range is about from0.25 to 25 mg as a single dose or in divided doses. Suitable dailydosages for patients are on the order of from e.g. 0.2 to 25 mg p.o.preferably 5 to 25. The compounds of formula I may be administered byany conventional route, in particular enterally, e.g. orally, e.g. inthe form of tablets, capsules, drink solutions, nasally, pulmonary (byinhalation) or parenterally, e.g. in the form of injectable solutions orsuspensions. Suitable unit dosage forms for oral administration comprisefrom ca. 0.05 to 12.5 mg, usually 1 to 10 mg active ingredient, e.g.Compound A, together with one or more pharmaceutically acceptablediluents or carriers therefor.

When used to prevent or treat chronic rejection or xenotransplantrejection as hereinabove specified the compounds of formula 1 may beadministered as the sole active ingredient or together with other drugsin immunomodulating regimens. For example, the compounds of formula Imay be used in combination with cyclosporins or ascomycins, or theirimmunosuppressive analogs, e.g. cyclosporin A, cyclosporin G, FK-506,etc.; corticosteroids; cyclophosphamide; azathioprene; methotrexate;brequinar; leflunomide; mizoribine; mycophenolic acid; mycophenolatemofetil; 15-deoxyspergualine, immunosuppressive monoclonal antibodies,e.g., monoclonal antibodies to leukocyte receptors, e.g., MHC, CD2, CD3,CD4, CD7, CD25, CD28, B7, CD45, or CD58 or their ligands; or otherimmunomodulatory compounds, e.g. CTLA4Ig.

Where the compounds of formula I are administered in conjunction withother immuno-suppressive/immunomodulatory therapy, e.g. for preventingor treating chronic rejection or xenotransplant rejection as hereinabovespecified, dosages of the co-administered immunosuppressant orimmuno-modulatory compound will of course vary depending on the type ofco-drug employed, e.g. whether it is a steroid or a cyclosporin, on thespecific drug employed, on the condition being treated, and so forth. Inaccordance with the foregoing the present invention provides in a yetfurther aspect:

8. A method as defined above comprising co-administration, e.g.concomitantly or in sequence, of a therapeutically effective amount of acompound of formula I and a second drug substance, said second drugsubstance being an immunosuppressant or immunomodulatory drug, e.g. asindicated above.

Formulation Example: capsules

Ethanol 20.0 mg 1,2-propylene glycol 81.0 mg Refined oil 121.5 mgCremophor RH40 202.5 mg Compound A 20.0 mg Total 500 mg

Compounds of formula I are well tolerated at dosages required for use inaccordance with the present invention. For example, the NTEL forCompound A in a 4-week toxicity study is 0.5 mg/kg/day in rats and 1.5mg/kg/day in monkeys.

1. A method for inhibiting or treating vascular surgery inducedneointimal proliferation and thickening and/or restenosis and/orvascular occlusion comprising administering to a subject in need thereofan effective amount of 40-O-(2-hydroxy)ethyl-rapamycin, wherein saidmethod excludes co-administering one or more active pharmaceuticalagents in addition to the effective amount of40-O-(2-hydroxy)ethyl-rapamycin.
 2. A method according to claim 1 forinhibiting or treating neointimal proliferation and thickening.
 3. Amethod according to claim 1 for inhibiting or treating restenosis and/orvascular occlusion.
 4. A method according to claim 1 for inhibiting ortreating vascular occlusion.
 5. A method according to claim 1, whereinthe vascular surgery is angioplasty.
 6. A method for inhibiting vascularsurgery induced neointimal proliferation and thickening and/orrestenosis and/or vascular occlusion comprising administering to asubject in need thereof an effective amount of40-O-(2-hydroxy)ethyl-rapamycin, wherein said method excludesco-administering one or more active pharmaceutical agents in addition tothe effective amount of 40-O-(2-hydroxy)ethyl-rapamycin.
 7. A methodaccording to claim 6 for inhibiting neointimal proliferation andthickening.
 8. A method according to claim 6 for inhibiting restenosisand/or vascular occlusion.
 9. A method according to claim 6 forinhibiting vascular occlusion.
 10. A method according to claim 6,wherein the vascular surgery is angioplasty.
 11. A method for treatingvascular surgery induced neointimal proliferation and thickening and/orrestenosis and/or vascular occlusion comprising administering to asubject in need thereof an effective amount of40-O-(2-hydroxy)ethyl-rapamycin, wherein said method excludesco-administering one or more active pharmaceutical agents in addition tothe effective amount of 40-O-(2-hydroxy)ethyl-rapamycin.
 12. A methodaccording to claim 11 for treating neointimal proliferation andthickening.
 13. A method according to claim 11 for treating restenosisand/or vascular occlusion.
 14. A method according to claim 11 fortreating vascular occlusion.
 15. A method according to claim 11, whereinthe vascular surgery is angioplasty.